Confocal microscope

ABSTRACT

A confocal microscope comprises a light source emitting a polarized light beam, an objective lens irradiating the polarized light beam, which is deflected and scanned by the optical scanner, to the sample as an excitation light beam, a wavelength separator detecting a necessary wavelength band from a polarized fluorescence emitted from the sample which is excited by the polarized light beam, and a photodetector unit having a polarization property extractor extracting a fluorescence with a predetermined polarization property from the fluorescence detected with the wavelength separator, a wavelength selector selecting a wavelength of the fluorescence extracted by the polarization property extractor, and a photodetector detecting the fluorescence selected by the wavelength selector.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based upon and claims the benefit of priorityfrom the prior Japanese Patent Applications No. 2002-381492, filed Dec.27, 2002; and No. 2003-314402, filed Sep. 5, 2003, the entire contentsof both of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a confocal microscope whichexcites a sample labeled with a fluorescence dyestuff and a fluorescentprotein by use of an excitation wavelength, and detects a fluorescenceemitted from the sample.

[0004] 2. Description of the Related Art

[0005] Heretofore, as a confocal microscope, a multi-color confocalmicroscope in which a multi-wavelength fluorescence detection system isemployed has been known (e.g., see U.S. Pat. No. 5,127,730).

[0006] The multi-color confocal microscope irradiates a sample havingpositions differently dyed with two or more fluorescent colorants withlaser beams having wavelengths corresponding to the respectivefluorescent colorants, and then detects fluorescent wavelengths, atwhich excitation occurs to generate the fluorescence, through wavelengthseparating means such as a dichroic mirror for these fluorescentwavelengths.

[0007] A confocal microscope capable of estimating a polarizingdirection of the fluorescence dyestuff has also been known. For example,in the confocal microscope disclosed in Jpn. Pat. Appln. KOKAIPublication No. 8-254654, a sample is irradiated with laser beamsemitted from a laser light source through an objective lens, and thenthrough the objective lens, the fluorescence emitted from the sample isbranched into two optical paths by the dichroic mirror. Afterward, thebranched beams are passed through polarizers which cross with each otherat right angles, to obtain two images in accordance with thepolarization of the fluorescence dyestuff.

[0008] In recent years, for example, when a target protein of livingcells is labeled with a fluorescence to observe distribution or movementthereof, a fluorescent protein such as GFP (green fluorescent protein)has often been utilized as a marker tracer.

[0009] A sample labeled with such GFP has polarization properties asdescribed in BIOPHOTONICS International May, 2002, p. 10. Thus, bydetecting the fluorescence polarization of the sample using afluorescent protein such as GFP, it becomes possible to analyzemolecular movement of the protein and a fluorescent life. Moreover, amolecular structure of the fluorescent protein changes by lightstimulation, chemical reaction, electrical stimulation, pH andtemperature variation or the like, so that the polarization propertieschange. Therefore, the analysis of the polarization properties of thefluorescent protein enables the analysis of a function of the protein.

[0010] Additionally, in the U.S. Pat. No. 5,127,730, only a generaldetecting method of a multi-wavelength fluorescence is disclosed, andthe detection of a fluorescence having polarized components is notdescribed. Moreover, in Jpn. Pat. Appln. KOKAI Publication No. 8-254654,it is described that two images are acquired in accordance withpolarization of a fluorescence dyestuff by the fluorescence from thesample passed through the polarizers which cross with each other atright angles, but only the confocal microscope which emits a wavelengthlight to generate the fluorescence is described. Moreover, an excitationmethod for obtaining the fluorescence having the polarized componentswith the use of the fluorescent proteins such as GFP as the fluorescencelabel is not described in the Jpn. Pat. Appln. KOKAI Publication No.8-254654.

BRIEF SUMMARY OF THE INVENTION

[0011] A confocal microscope according to the first aspect of thepresent invention is characterized by comprising: a light sourceemitting a polarized light beam; an objective lens irradiating thepolarized light beam, which is deflected and scanned by the opticalscanner, to the sample as an excitation light beam; a wavelengthseparator detecting a necessary wavelength band from a polarizedfluorescence emitted from the sample which is excited by the polarizedlight beam; and a photodetector unit having a polarization propertyextractor extracting a fluorescence with a predetermined polarizationproperty from the fluorescence detected with the wavelength separator, awavelength selector selecting a wavelength of the fluorescence extractedby the polarization property extractor, and a photodetector detectingthe fluorescence selected by the wavelength selector.

[0012] A confocal microscope according to the second aspect of thepresent invention is characterized by comprising: a light sourceemitting a polarized light beam; an objective lens condensing thepolarized light beam on a sample; a rotational disk having a pluralityof pinholes or slits and leading the polarized light beam from the lightsource to the objective lens, a fluorescence image emitted from thesample being projected on the rotational disk through the objectivelens; a wavelength separator detecting a necessary wavelength band froman image passing the rotational disk; a polarization property extractorextracting a fluorescence with a predetermined polarization propertyfrom the fluorescence detected with the wavelength separator; and animaging unit imaging the fluorescence extracted by the polarizationproperty extractor.

[0013] A confocal microscope according to the third aspect of thepresent invention is characterized by comprising: a light sourceemitting a beam; a polarizer polarizing the light beam; an opticalscanner deflecting and scanning the polarized light beam; an objectivelens irradiating the polarized light beam, which is deflected andscanned by the optical scanner, to the sample as an excitation lightbeam; a wavelength separator detecting a necessary wavelength band froma polarized fluorescence emitted from the sample which is excited by thepolarized light beam; and a photodetector unit having a polarizationproperty extractor extracting a fluorescence with a predeterminedpolarization property from the fluorescence detected with the wavelengthseparator, a wavelength selector selecting a wavelength of thefluorescence extracted by the polarization property extractor, and aphotodetector detecting the fluorescence selected by the wavelengthselector.

[0014] Advantages of the invention will be set forth in the descriptionwhich follows, and in part will be obvious from the description, or maybe learned by practice of the invention. Advantages of the invention maybe realized and obtained by means of the instrumentalities andcombinations particularly pointed out hereinafter.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0015] The accompanying drawings, which are incorporated in andconstitute a part of the specification, illustrate embodiments of theinvention, and together with the general description given above and thedetailed description of the embodiments given below, serve to explainthe principles of the invention.

[0016]FIG. 1 is a diagram showing a schematic configuration of a firstembodiment of the present invention;

[0017]FIG. 2 is a diagram showing the schematic configuration ofModification 1 of the first embodiment;

[0018]FIG. 3 is a diagram showing the schematic configuration ofModification 2 of the first embodiment;

[0019]FIG. 4 is a diagram showing the schematic configuration of asecond embodiment of the present invention;

[0020]FIG. 5 is a diagram showing the schematic configuration of thesecond embodiment;

[0021]FIG. 6 is a diagram showing the schematic configuration of themodification of the second embodiment;

[0022]FIG. 7 is a diagram showing the schematic configuration of a thirdembodiment of the present invention; and

[0023]FIG. 8 is a diagram showing the schematic configuration of afourth embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0024] Embodiments of the present invention will hereinafter bedescribed with reference to the drawings.

[0025] (First Embodiment)

[0026]FIG. 1 shows a schematic configuration of a confocal laserscanning microscope to which the present invention is applied.

[0027] In FIG. 1, a laser light source 1 emits a pulsed laser beamhaving polarized components.

[0028] A polarizer 2, a dichroic mirror 3 used as wavelength dividingmeans, and a scanning optical unit 4 use as light scanning means arearranged on an optical path of the laser beam outputted from the laserlight source 1.

[0029] The polarizer 2 is used to improve and optimize polarizationproperties (extinction ratio) of the laser light source 1, and the laserlight source 1 and the polarizer 2 constitute light source means whichhave polarization properties. The dichroic mirror 3 has properties fordetecting a necessary wavelength band, transmits an incident laser beamfrom the laser light source 1, and reflects (detects) a fluorescencefrom the scanning optical unit 4. The scanning optical unit 4 includesscanning mirrors 4 a, 4 b, and the scanning mirrors 4 a, 4 b deflect thelaser beam.

[0030] A relay lens 5 and a mirror 6 are arranged on the optical path ofthe laser beam deflected by the scanning optical unit 4. An imageformation lens 7 and an objective lens 8 are arranged on a reflectedoptical path of the mirror 6.

[0031] A sample 10 laid on a stage 9 is irradiated with the laser beamreflected by the mirror 6 and passed through the image formation lens 7.The light with which a section 10 a of the sample 10 is scanned in apredetermined range on the section 10 a by movement. of the scanningmirrors 4 a, 4 b.

[0032] Fluorescent proteins such as GFP are used as fluorescence labelsto the sample 10. The sample 10 is excited by the laser beam focused ona focal position and having polarized components to absorb the light ina polarizing direction in a transition moment of fluorescence moleculeswhich agrees with the polarizing direction, and is brought into anexcited state. In this case, the fluorescence deactivated from theexcited state also forms a polarized light which agrees with thetransition moment.

[0033] Accordingly, the fluorescence (hereinafter referred to as apolarized fluorescence) having the polarized components is generatedfrom the sample 10. This polarized fluorescence is collected on theobjective lens 8, passed through the image formation lens 7, andincident upon the dichroic mirror 3 through the mirror 6, relay lens 5,and scanning optical unit 4.

[0034] A polarizer 11 which is use as polarization property extractionmeans constituting photodetection means, a barrier filter 12 which useas is wavelength selection means, a confocal lens 13, a confocal pinhole14, and a photomultiplier 15 which is use as a photodetection unit, arearranged on a reflected optical path deflected by 90 degrees by thedichroic mirror 3.

[0035] The polarizer 11 extracts the polarized fluorescence which haspredetermined polarization properties. The barrier filter 12 selects awavelength of the polarized fluorescence. The image of the polarizedfluorescence selected from the barrier filter 12 is formed on a confocalpinhole 14 plane through the confocal lens 13. The polarizedfluorescence passed through the confocal pinhole 14 is detected by thephotomultiplier 15.

[0036] In this case, the same effect is obtained, even when thepolarizer 11 is disposed after the confocal lens 13 and confocal pinhole14. As the polarizer 11, a polarized beam splitter (PBS), ½ wavelengthplate, polarizing rotator, liquid crystal shutter, Pockel cell, and thelike may be used as long as the polarizing direction can be detected.Here, with the PBS, the polarized fluorescence can be split into a Ppolarized light and S polarized light. With the ½ wavelength plate, thepolarizing direction can be changed. Therefore, when an AO device havingthe same function as that of the barrier filter 12 is used, the light isinserted in accordance with the polarizing direction of AO, and it ispossible to select the wavelength by the AO.

[0037] Next, an operation of an embodiment constituted in this mannerwill be described.

[0038] When the laser beam having the polarized components is emittedfrom the laser light source 1, the laser light polarization propertiesare optimized by the polarizer 2, transmitted through the dichroicmirror 3, and incident upon the scanning optical unit 4. The laser beamincident upon the scanning optical unit 4 is deflected by the scanningmirrors 4 a, 4 b.

[0039] The laser beam deflected by the scanning optical unit 4 isincident upon the image formation lens 7 through the relay lens 5 andmirror 6. The laser beam passed through the image formation lens 7 isfocused on the section 10 a of the sample 10 laid on the stage 9.

[0040] The sample 10 is excited by the polarized laser beam focused onthe focal position to absorb the light in the polarizing direction inthe transition moment of the fluorescence molecules which agrees withthe polarizing direction, and is brought into the excited state. In thiscase, the fluorescence deactivated from the excited state also forms thepolarized light which agrees with the transition moment.

[0041] The polarized fluorescence emitted from the sample 10 is incidentupon the dichroic mirror 3 through the objective lens 8, image formationlens 7, mirror 6, relay lens 5, and scanning optical unit 4 in adirection reverse to the previous optical path.

[0042] The polarized fluorescence deflected by the dichroic mirror 3 by90 degrees is incident upon the polarizer 11. The polarizer 11 extractsthe fluorescence having predetermined polarization properties to guidethe fluorescence into the barrier filter 12. The barrier filter 12selects only the predetermined wavelength of the polarized fluorescence,and forms the image on the confocal pinhole 14 plane through theconfocal lens 13. The polarized fluorescence passed through the confocalpinhole 14 is incident upon the photomultiplier 15. The photomultiplier15 detects luminance of the incident polarized fluorescence, andconverts it into an electric signal to output polarized fluorescenceconfocal image data.

[0043] Therefore, in this case, when the sample 10 labeled with thefluorescent proteins such as GFP is irradiated with the laser beamhaving the polarized components as an excited light, the sample 10 cansecurely be excited.

[0044] Moreover, the polarized fluorescence generated from the sample 10by the excitation is detected via the dichroic mirror 3 which is used aswavelength dividing means, the polarizer 11 which is used as thepolarization property extraction means, and the barrier filter 12 whichis used as wavelength selection means. Accordingly, molecular movementof the protein and a fluorescent life concerning the polarizedcomponents can be analyzed from data of the polarized fluorescence. Inthis case, a molecular structure of the fluorescent protein changes bylight stimulation, chemical reaction, electrical stimulation, pH andtemperature variation or the like, so that polarization propertieschange. Therefore, the analysis of the polarization properties of thefluorescent protein enables the analysis of various functions of theprotein.

[0045] Furthermore, to analyze the fluorescent proteins such as GFP, aspecific portion in the cell is sometimes labeled. However, when thepolarized components differ with a thickness direction of the sample inthis manner, the polarized components of a portion other than a portionto be detected are synthesized in a general microscope, and it isdifficult to detect the polarized components of the portion.Additionally, in the embodiment, since the specific portion of a samplein the thickness direction can be detected by a sectioning effect of theconfocal microscope, it is possible to securely obtain information ofthe specific portion in the cell in the thickness direction.

[0046] It is to be noted that with the use of an IR pulse laser as thelaser light source 1, a polarized fluorescence image can be acquired bytwo-photon absorption. Since a two-photon absorption phenomenon occursonly in an image formation position in this case, the confocal pinhole14 is conceptually unnecessary. The dichroic mirror 3 for use in thiscase has short-wavelength reflection properties that the IR laser istransmitted, and a visible polarized fluorescence is reflected andguided on the side of the photomultiplier 15.

[0047] (Modification 1)

[0048] An example of detection of the polarized fluorescence of thesample 10 has been described in the first embodiment, but it is alsopossible to measure the fluorescent life. In FIG. 2, the same componentsas those of FIG. 1 are denoted with the same reference numerals.

[0049] In this case, a half mirror 20 is inserted as light dividingmeans in the reflected optical path of the dichroic mirror 3. Moreover,the above-described polarizer 11, barrier filter 12, confocal lens 13,confocal pinhole 14, and photomultiplier 15 are arranged in atransmission optical path of the half mirror 20, and a polarizer 21,barrier filter 22, confocal lens 23, confocal pinhole 24, andphotomultiplier 25 are arranged in the reflected optical path.

[0050] Here, assuming that the polarizer 11 and the polarizer 21 extracts-polarized components and p-polarized components of polarizedfluorescence, respectively, fluorescent intensities of the s-polarizedand p-polarized components extracted by the polarizers 11, 21 aredetected by the is photomultipliers 15, 25.

[0051] Moreover, the photomultipliers 15, 25 are connected to a personalcomputer (PC) 26 which is calculation means. The PC 26 calculates ananisotropy ratio r(t) based on the fluorescent intensities of thes-polarized and p-polarized components detected by the photomultipliers15, 25 by the following equation.

r(t)=[Is(t)−Ip(t)]/[Is(t)+2·Ip(t)]  (1),

[0052] where Is(t) denotes the fluorescent intensity of the s-polarizedcomponent, and Ip(t) denotes the fluorescent intensity of thep-polarized component.

[0053] The anisotropy ratio r(t) has a certain relation with rotationrelaxation time and oscillation diffusion speed, and various dynamicproperties of molecules can be known by the anisotropy ratio r(t). WhenIs(t)+Ip(t) is further obtained, the fluorescent life of the fluorescentprotein can also be obtained from an attenuation curve regardless of arotational movement. The dynamic properties and fluorescent life of themolecules and fluorescent protein also change depending on varioussurrounding conditions. Therefore, these information can simultaneouslybe measured, the information can be an important bioscientific analysistool.

[0054] (Modification 2)

[0055] An example in which one laser light source and one photodetectionsection are arranged has been described in the first embodiment but, asshown in FIG. 3, two laser light sources and two photodetection sectionsmay also be disposed. In FIG. 3, the same components as those of FIG. 1are denoted with the same reference numerals.

[0056] In Modification 2, a laser light source 31 is disposed togetherwith the laser light source 1. The laser light source 31 emits the laserbeam having the polarized components different from the laser lightsource 1 in wavelength.

[0057] A synthesis mirror 32 is disposed via the polarizer 2 on theoptical path of the laser beam outputted from the laser light source 1.A mirror 34 is disposed via a polarizer 33 on the optical path of thelaser beam outputted from the laser light source 31. The laser beamreflected by the mirror 34 is incident upon the synthesis mirror 32.

[0058] The synthesis mirror 32 can be replaced with the dichroic mirrorhaving wavelength properties or the polarized beam splitter (PBS) havingpolarization properties. Acoustic optical devices such as AOTF can alsobe used to synthesize the light.

[0059] The dichroic mirror 3 and scanning optical unit 4 are arrangedvia a polarizer 35 on the optical path of the laser beam synthesized bythe synthesis mirror 32.

[0060] On the other hand, a half mirror 37 is inserted as light dividingmeans via a polarizer 36 on the reflected optical path of the dichroicmirror 3. Moreover, the polarizer 11, barrier filter 12, confocal lens13, confocal pinhole 14, and photomultiplier 15 are arranged as firstphotodetection means on the transmission optical path of the half mirror37, and a polarizer 38, barrier filter 39, confocal lens 40, confocalpinhole 41, and photomultiplier 42 are arranged on the reflected opticalpath.

[0061] When a plurality of laser light sources 1, 31 and photodetectionmeans 43, 44 are prepared in this manner, a sample of a multi-wavelengthfluorescence or a sample partially different in the polarizing directioncan be handled.

[0062] Here, to handle the sample 10 of the multi-wavelengthfluorescence, the wavelengths of the laser light sources 1, 31 are setfor the fluorescence wavelengths, and the wavelength is selected and setby the barrier filters 12, 39 which are wavelength selection means foreach of the photodetection means 43, 44. To handle the sample partiallydifferent in the polarizing direction, the laser light sources 1, 31different in the polarization properties are prepared, and thepolarizing directions of the laser light sources 1, 31 may be selectedfor each of portions having different polarizing directions on thesample.

[0063] It is to be noted that in the modification, an example in whichtwo laser light sources and two. photodetection means are arranged hasbeen described, and two or more laser light sources and photodetectionmeans may also be disposed.

[0064] (Second Embodiment)

[0065] Next, a second embodiment of the present invention will bedescribed. The same components as those of FIG. 1 are denoted with thesame reference numerals.

[0066]FIG. 4 is a diagram showing the schematic configuration of thesecond embodiment. The same components as those of FIG. 1 are denotedwith the same reference numerals.

[0067] In the second embodiment, an input end 511 of an optical fiber 51is disposed on the optical path of the laser beam outputted from thelaser light source 1. The optical fiber 51 transmits the laser beamhaving the polarized components from the laser light source 1. Forexample, a polarized wave plane storage fiber is preferably used as theoptical fiber 51.

[0068] A rotating mechanism 52 is disposed as polarizing directionchanging means in an output end 512 of the optical fiber 51. Therotating mechanism 52 is capable of rotating the output end 512 of theoptical fiber 51, and the polarizing direction of the laser beam isfreely changed in accordance with a rotational angle of the output end512. That is, the rotating mechanism 52 changes the polarizing directionof the laser beam in accordance with the polarizing direction of thesample 10. Accordingly, when the rotating mechanism 52 is rotated by 90degrees, the data of the polarized fluorescence by the excitation of thes-polarized and p-polarized components can selectively be acquired.

[0069] The polarizer 2 is disposed on the front surface of the rotatingmechanism 52. This polarizer 2 can be rotated in accordance with therotation of the rotating mechanism 52, and the polarizing direction isthe same as that of the light emitted from the rotating mechanism 52.

[0070] The rotating mechanism 52 is connected to a controller 53 whichis use as control means. The controller 53 is connected to the scanningoptical unit 4 and photomultiplier 15, and is further connected to anoperation unit 54 and monitor 55.

[0071] The controller 53 rotates/controls the rotating mechanism 52 bythe operation of the operation unit 54, and the polarizing direction ofthe laser beam can arbitrarily be set. The controller 53 includes means(not shown) for detecting each portion on the sample 10 based onscanning information of the scanning optical unit 4 in accordance withthe movement of the scanning mirrors 4 a, 4 b on the sample 10, and therotating mechanism 52 is rotated/controlled by detected informationhere. Moreover, the image information of each portion on the sample 10is displayed on the monitor 55 by the data of the photomultiplier 15acquired in this manner.

[0072] In this case, the rotating mechanism 52 is rotated by thecontroller 53 in accordance with the polarizing direction of the sample10, and the polarizing direction of the laser beam can be changed toselectively acquire the information in accordance with the polarizingdirection by the excitation of the s-polarized and p-polarizedcomponents on the sample 10.

[0073] Moreover, when it is known that the characteristics of thepolarizing direction etc. on the sample 10 differs with each portion,for example, as shown in A (s-polarized), B (p-polarized), C(no-polarized) in FIG. 5, the rotating mechanism 52 is rotated based onthe scanning information of the scanning optical unit 4 on the sample 10in accordance with the movement of the scanning mirrors 4 a, 4 b, thatis, in accordance with each portion of A, B, C on the sample 10 tochange the polarizing direction of the laser beam. Accordingly, imageinformation of each portion on the sample 10 can be displayed on themonitor 55 (FIG. 5).

[0074] Furthermore, when the optical fiber 51 is used to introduce thelaser beam outputted from the laser light source 1 into the rotatingmechanism 52 here, a rotating portion of the rotating mechanism 52 canbe compact. In this case, when the light having different wavelength andthe laser beam having different polarizing direction are synthesized andintroduced on the side of the input end 511 of the optical fiber 51, thepolarizing directions of a large number of laser beams can be changedwith one rotating mechanism 52.

[0075] It is to be noted that in the second embodiment, in a method ofchanging the polarizing direction of the laser beam, a mechanicalconfiguration such as the rotating mechanism 52 has been used, butanother method may also be used as long as the polarizing direction canbe changed. For example, a method of using an optical polarizing rotatoris used. Alternatively, a laser for random polarization is used in thelight source, and a ¼ wavelength plate may also be combined with thepolarizer to extract and use arbitrary polarized components from therandom light. In this case, a ½ wavelength plate may also be combined tochange the polarization to p-polarization and s-polarization.Furthermore, the whole laser light source 1 may be rotated around anoptical axis to change the polarizing direction of the laser beam.

[0076] (Modification)

[0077] In the second embodiment, an example in which one laser lightsource and one photodetection means are disposed has been described. Twolaser light sources and two photodetection means may also be disposed asshown in FIG. 6 in which the same components as those of FIG. 4 aredenoted with the same reference numerals.

[0078] In this case, a laser light source 60 is disposed together withthe laser light source 1. The laser light source 60 emitting the laserbeam having the polarized components in the same manner as in the laserlight source 1 is used.

[0079] A synthesis mirror 62 is disposed via an optical polarizingrotator 61 on the optical path of the laser beam outputted from thelaser light source 1. A mirror 64 is disposed via an optical polarizingrotator 63 on the optical path of the laser beam outputted from thelaser light source 60. Here, the optical polarizing rotators 61, 63 arecapable of arbitrarily setting the polarizing direction of the laserbeam of the laser light sources 1, 60.

[0080] The laser beam reflected by the mirror 64 is incident upon thesynthesis mirror 62. The synthesis mirror 62 can be replaced with thedichroic mirror having the wavelength properties or the polarized beamsplitter (PBS) having the polarization properties. The acoustic opticaldevices such as AOTF can also be used to synthesize the light.

[0081] The input end 511 of the optical fiber 51 is disposed on theoptical path of the laser beam synthesized by the synthesis mirror 62.The dichroic mirror 3 and scanning optical unit 4 are arranged via apolarizer 65 on the side of the output end 512 of the optical fiber 51.

[0082] On the other hand, a half mirror 67 is inserted as the lightdividing means via a polarizer 66 on the reflected optical path of thedichroic mirror 3. The polarizer 11, barrier filter 12, confocal lens13, confocal pinhole 14, and photomultiplier 15 are arranged as firstphotodetection means 73 in the transmission opticalpath of the halfmirror 67. A polarizer 68, barrier filter 69, confocal lens 70, confocalpinhole 71, and photomultiplier 72 are arranged as second photodetectionmeans 74 in the reflected optical path.

[0083] In this manner, the polarizing rotators 61, 63 may be operatedwith respect to the laser light sources 1, 60 to individually set thepolarizing directions of the laser beam. That is, the polarizing rotator61 may be operated to s-polarize the laser beam of the laser lightsource 1, the polarizing rotator 63 may be operated to p-polarize thelaser beam of the laser light source 60, and the s-polarized andp-polarized laser beams may be synthesized and given as an excited lightto the sample 10. Needless to say, the polarizing rotator 61 may beoperated to p-polarize the laser beam of the laser light source 1, andthe polarizing rotator 63 may also be operated to s-polarize the laserlight of the laser light source 60.

[0084] Accordingly, even when the polarizing direction differs with eachportion of the sample 10, the polarizing direction of the laser beam ofthe laser light sources 1, 60 can be set in an optimum state inaccordance with the difference of the polarizing direction. In thiscase, the polarized 65 is removed or synchronously rotated.

[0085] Moreover, two laser light sources 1, 60 can be used/applied in acase where the wavelength of the laser beam differs in order to excitethe different fluorescence wavelength and in a case where the wavelengthof the laser beam is the same and the polarizing direction differs. Whenthe wavelength of the laser beam differs, the laser beam can be selectedand used with respect to the fluorescence wavelength of the sample 10 tohandle a multi-wavelength fluorescence sample. When the wavelength ofthe laser beam is the same and the polarizing direction is different, bychanging the polarizing direction with the polarizing rotators 61, 63,and the sample 10 different in the polarization properties can behandled for each fluorescence wavelength.

[0086] It is to be noted that an example in which two laser lightsources and two photodetection means are arranged has been describedabove, but two or more laser light sources and photodetection means mayalso be arranged.

[0087] (Third Embodiment)

[0088] A third embodiment of the present invention will next bedescribed.

[0089] In the first and second embodiments, the confocal laser scanningmicroscope in which the laser beam is focused on a spot and scanned hasbeen described. The confocal microscope has another system, and it ispossible to obtain the similar effect.

[0090]FIG. 7 shows the schematic configuration of the third embodiment.

[0091] In FIG. 7, the light emitted from a light source 81 is formedinto a parallel light by a collimator lens 82, and is incident upon apolarizer 83. The polarizer 83 converts the light of the light source 81to the light which has the polarization properties. The polarizer 83constitutes light source means which has the polarization propertiestogether with the light source 81.

[0092] The light incident upon the polarizer 83 is converted to thelight which has the polarization properties, and the light having anexcited wavelength width is selected by a wavelength separator 84. Here,the dichroic mirror, AOM, and the like are used in the wavelengthseparator 84.

[0093] The light selected by the wavelength separator 84 is passedthrough a rotational disk 85, and is incident upon a focal position on asample 88 surface as an excited light via an image formation lens 86 andobjective lens 87.

[0094] The rotational disk 85 has a function of regulating the lightwith a pinhole or a slit etc. having an airy diameter of the objectivelens 87 or the airy diameter×about 0.5. The rotational disk 85 isdisposed on a focal plane which is a position optically conjugated withthe objective lens 87, and is connected to a shaft of a motor (notshown) via a rotation shaft 851 to rotate at a certain rotation speed.

[0095] The sample 88 generates the fluorescence having the polarizedcomponents by the excited light, and projects a fluorescence image onthe rotational disk 85 by the image formation lens 86 via the objectivelens 87. A focused portion of the projected image is passed through thepinhole or slit to obtain a confocal effect. The portion is furtherpassed through the wavelength separator 84, and the polarized componentsare selected by a polarizer 89, and thereafter imaged by a CCD camera 91which is imaging means via an image formation lens 90.

[0096] It is possible to obtain the same effect as that of theabove-described confocal laser scanning microscope by the confocalmicroscope constituted in this manner. In the confocal microscope, awhite light source, LED, laser light source, and the like may be used inthe light source 81. When the laser light source is used, the laserlight source having the polarized components may be used to omit thepolarizer 83.

[0097] (Fourth Embodiment)

[0098] Next, a fourth embodiment of the present invention will bedescribed.

[0099] In the fourth embodiment, the present invention will furtherconcretely be described. FIG. 8 shows the schematic configuration of theconfocal laser scanning microscope to which the fourth embodiment isapplied.

[0100] In FIG. 8, a laser light source 11 generates a pulsed laser beamhaving the polarized components as an excited light. In this case, asmall-sized semiconductor laser in which the laser beam is easily turnedon/off is used in the laser light source 101.

[0101] A capacitor lens 102, a polarizer 103, and a dichroic mirror 104are arranged on the optical path of the laser beam from the laser lightsource 101.

[0102] The capacitor lens 102 collimates the excited light from thelaser light source 101 in an optimum diameter. The polarizer 103 hasproperties for improving and optimizing the polarization properties(extinction ratio) of the laser light source 1, and the dichroic mirror104 has properties for detecting a necessary wavelength band. Thedichroic mirror 104 reflects the laser beam incident from the laserlight source 101, and transmits (detects) the fluorescence incident onthe side of a scanning optical unit 105. It is to be noted that thedichroic mirror 104 is detachably attached so as to change thecorresponding properties, when the wavelength of the excited light orthe wavelength of the fluorescence emitted from a sample 110 describedlater is changed if necessary.

[0103] A scanning optical unit 105 is disposed in the reflected opticalpath of the dichroic mirror 104. The scanning optical unit 105 includesscanning mirrors 105 a, 105 b, and the laser beam is deflected by thesescanning mirrors 105 a, 105 b.

[0104] A pupil projection lens 106 and mirror 107 are arranged in theoptical path of the laser beam deflected by the scanning optical unit105. An image formation lens 108 and an objective lens 109 are arrangedin the reflected optical path of the mirror 107.

[0105] The laser beam reflected by the mirror 107 and passed through theimage formation lens 108 is scanned over an entire view field of theobjective lens 109 by the movement of the scanning mirrors 105 a, 105 b.

[0106] Also in this case, the fluorescent proteins such as GFP are usedas the fluorescent labels in the sample 110. The sample 110 is excitedby the laser beam having the polarized components focused in the focalposition, and absorbs the light in the polarizing direction in thetransition moment of the fluorescent molecules which agrees with thepolarizing direction, and is brought into the excited state. In thiscase, the fluorescence deactivated from the excited state also forms apolarized light which agrees with the transition moment.

[0107] Accordingly, the fluorescence (hereinafter referred to as“polarized fluorescence”) having the polarized components is generatedfrom the sample 110. The polarized fluorescence is focused on theobjective lens 109, passed through the image formation lens 108, andincident upon the dichroic mirror 104 through the mirror 107, pupilprojection lens 106, and scanning optical unit 105. The dichroic mirror104 separates a return light in which the polarized fluorescence ismixed with the excited light, and transmits the polarized fluorescence.

[0108] A condensing lens 111 and confocal pinhole 112 are arranged inthe transmission optical path of the dichroic mirror 104. The condensinglens 111 forms the polarized fluorescence emitted from one point of thesample 110 into the image on the confocal pinhole 112. The confocalpinhole 112 is disposed in a position optically conjugated with a focalpoint of the objective lens 109, and transmits focused components in thepolarized fluorescence from the sample 110, but cannot transmitnon-focused components. In this case, the size of the confocal pinhole112 needs to be smaller than that of the airy disk formed by thecondensing lens 111 in order to sufficiently realize a confocal effect.Therefore, when the objective lens 109 is changed, a mechanism isaccordingly preferably disposed in which the size of the pinhole ischanged to a different size. Concretely, for example, a disc-shapedturret including a pinhole having a different size may be rotated insynchronization with a revolver (not shown) for use in switching theobjective lens 109.

[0109] A polarized beam splitter 113 is disposed on the optical path ofthe light which comes out of the confocal pinhole 112. The polarizedbeam splitter 113 splits the light passed through the confocal pinhole112 into two polarized components crossing at right angles to eachother, that is, the p-polarized and s-polarized components. In thiscase, since the type of the fluorescence wavelength is various, a bandof the polarized beam splitter 113 is preferably as broad as possible.If possible, the polarized beam splitter is preferably detachablyattached in the same manner as in the dichroic mirror 104, so that thecharacteristics can be changed to the corresponding characteristics,when the wavelength of the fluorescence is changed.

[0110] A barrier filter 114 a, analyzer 115 a, and photodetection unit116 a are arranged as a first detection system in one optical path splitby the polarized beam splitter 113, and a barrier filter 114 b, analyzer115 b, and photodetection unit 116 b are arranged as a second detectionsystem in the other optical path. These two detection systems havesubstantially equal characteristics.

[0111] Here, the barrier filters 114 a, 114 b completely cut the excitedlight which cannot completely be cut off by the dichroic mirror 104.That is, in general, when a light emitting efficiency of fluorescence isnot very high, and especially when photons are counted by the pulselight excitation, the filters are used. Because an influence of the leakof the laser beam included in the fluorescence over fluorescencemeasurement is large as compared with another microscope observation.The analyzers 115 a, 115 b are used to realize correct measurement.Because both the transmitted light and the reflected light have a largeratio (1 to 5%) of mixture of unnecessary polarized components having anopposite direction, when the polarized beam splitter 113 is brought in abroader band. High-sensitivity and low-noise detecting units such as aphotomultiplier tube and an avalanche diode are used as thephotodetection units 116 a, 116 b.

[0112] It is to be noted that in FIG. 8, the light coming out of theconfocal pinhole 112 is drawn so as to spread largely. When a ratio of afocal distance between the condensing lens 111 and the pupil projectionlens 106 is increased, and an image formation magnification onto theconfocal pinhole 112 is raised, the spread of the light can sufficientlybe reduced with respect to the light receiving surfaces of thephotodetection units 116 a, 116 b. Needless to say, when there is asufficient space, an optical system for projecting the image of theconfocal pinhole 112 onto the photodetection units 116 a, 116 b may alsobe constituted.

[0113] On the other hand, a half mirror 116 is disposed between theimage formation lens 108 and the objective lens 109, and an observationlens tube 117 is disposed between the reflective mirror 107 and theimage formation lens 108.

[0114] An illuminative light from an observation illuminating unit 118is incident upon the half mirror 116. The illuminative light isreflected by the half mirror 116 to irradiate the sample 110 via theobjective lens 109. The reflected light from the sample 110 istransmitted through the half mirror 116, and is incident upon theobservation lens tube 117 via the image formation lens 108, so that asample image can be observed visually or on TV in a general opticalmicroscope.

[0115] Next, an operation of the embodiment constituted in this mannerwill be described.

[0116] When the pulsed laser beam is emitted from the laser light source101, the laser beam is collimated by the collimator lens 102, and thepolarization properties are optimized by the polarizer 103. Thereafter,the light is reflected by the dichroic mirror 104 and is incident uponthe scanning optical unit 105.

[0117] The laser beam incident upon the scanning optical unit 105 isdeflected by the scanning mirrors 105 a, 105 b, and is incident upon theimage formation lens 108 via the pupil projection lens 106 and mirror107. The laser beam transmitted through the image formation lens 108 iscondensed on the sample 110.

[0118] The sample 110 is brought into the excited state by the polarizedlaser beam focused in the focal position. In this case, the fluorescencedeactivated from the excited state also forms the polarized light whichagrees with the transition moment.

[0119] The polarized fluorescence emitted from the sample 110 isincident upon the dichroic mirror 104 through the objective lens 109,image formation lens 108, mirror 107, pupil projection lens 106, andscanning optical unit 105 in a direction opposite to that of theprevious optical path.

[0120] The polarized fluorescence transmitted through the dichroicmirror 104 is formed into the image on the confocal pinhole 112 throughthe condensing lens 111. The polarized fluorescence passed through theconfocal pinhole 112 is separated into two polarized components crossingat right angles to each other, that is, the p-polarized and s-polarizedcomponents by the polarized beam splitter 113.

[0121] The fluorescence of one polarized component separated by thepolarized beam splitter 113 is incident upon the photodetection unit 116a via the barrier filter 114 a and analyzer 115 a, and the fluorescenceof the other polarized component is incident upon the photodetectionunit 116 b via the barrier filter 114 b and analyzer 115 b. Thephotodetection units 116 a, 116 b detect luminance of the incidentfluorescence, converts the fluorescence into an electric signal, andoutputs confocal image data.

[0122] Moreover, an image of a rotation relaxation time of fluorescencemolecules can be obtained in consideration of a ratio or a differencewith respect to the polarized components of output signals from thephotodetection units 116 a, 116 b corresponding to the respectivescanning points of the sample 110 acquired in this manner. When a sum iscalculated, a fluorescent life image can be obtained regardless ofpresence/absence of molecular rotation. Furthermore, the number ofphotons is counted with the photodetection units 116 a, 116 b. When acoefficient of the total number of photons is obtained for each pixel,the fluorescent intensity image can be obtained. In this case, thenumber of excitation pulses emitted for each pixel has to be the same.

[0123] It is to be noted that a detection signal intensity (the numberof photons) with respect to the polarized components crossing at rightangles to one another in the fourth embodiment sometimes subtly differswith a transmittance of the polarized beam splitter 113, a difference ofreflectance, a difference of the transmittance between the barrierfilters 114 a, 114 b, a difference of the transmittance between theanalyzers 115 a, 115 b, and a difference of sensitivity between thephotodetection units 116 a, 116 b. When the above-described calculationis performed, this intensity needs to be incorporated as a correctioncoefficient to perform the calculation. When the dichroic mirror 104 isreplaced with that having appropriate wavelength properties, thepolarized beam splitter 113 is replaced with the half mirror, and bandpass filters having different wavelength band s are used instead of theanalyzers 115 a, 115 b, the fluorescent intensity image and fluorescentlife image having two different wavelengths can simultaneously beobtained.

[0124] Therefore, in this manner, a tomogram of a fluorescent intensitydistribution by the confocal effect can be acquired by one excited lightscanning with respect to the sample 110, and a rotation relaxation timeimage and fluorescent life image of labeled molecules can be acquired.When a small number of optical components are simply replaced, thedifference of properties on the sample by the fluorescence emittingvarious fluorescent wavelengths can be observed.

[0125] When the sample labeled with the fluorescent protein isirradiated with the laser beam having the polarized components as theexcited light according to the embodiment of the present invention, thesample can securely be excited. When the fluorescence having thepolarized components generated from the sample is detected via thewavelength dividing means, polarization property extracting means, andwavelength selection means, the molecular movement of the protein andthe fluorescent life can be analyzed from detected information.Furthermore, when the confocal microscope is combined, information oflocal polarized fluorescence properties in a cell can also be obtained.

[0126] Moreover, according to the embodiment of the present invention,since the polarizing direction of the laser beam can be changed inaccordance with the polarizing direction of the sample by polarizingdirection changing means, the data of the fluorescence having differentpolarized components on the sample can selectively be acquired.

[0127] Furthermore, since the polarizing direction of the polarizingdirection changing means can be controlled by the detected informationof each portion on the sample, the information can be displayed inaccordance with the polarizing direction of each portion on the sample.

[0128] According to the embodiment of the present invention, there canbe provided the confocal microscope in which the sample labeled with thefluorescent protein can be excited and various functions of the samplecan be analyzed by the polarized fluorescence obtained in this manner.

[0129] Additional advantages and modifications will readily occur tothose skilled in the art. Therefore, the present invention in itsbroader aspects is not limited to the specific details, representativedevices, and illustrated examples shown and described herein.Accordingly, various modifications may be made without departing fromthe spirit or scope of the general inventive concept as defined by theappended claims and their equivalents.

What is claimed is:
 1. A confocal microscope comprising: a light sourceemitting a polarized light beam; an objective lens irradiating thepolarized light beam, which is deflected and scanned by the opticalscanner, to the sample as an excitation light beam; a wavelengthseparator detecting a necessary wavelength band from a polarizedfluorescence emitted from the sample which is excited by the polarizedlight beam; and a photodetector unit having a polarization propertyextractor extracting a fluorescence with a predetermined polarizationproperty from the fluorescence detected with the wavelength separator, awavelength selector selecting a wavelength of the fluorescence extractedby the polarization property extractor, and a photodetector detectingthe fluorescence selected by the wavelength selector.
 2. The confocalmicroscope according to claim 1, wherein the light source has apolarizing direction changer changing a polarizing direction.
 3. Theconfocal microscope according to claim 2, wherein the polarizingdirection changer rotates the entire light source around a light axis ofthe light beam emitted from the light source.
 4. The confocal microscopeaccording to claim 2, further comprising an optical fiber transmittingthe polarized light beam emitted from the light source, wherein thepolarizing direction changer is provided to the optical fiber.
 5. Theconfocal microscope according to claim 4, wherein the polarizingdirection changer has a rotation mechanism capable of rotating an outputend portion of the optical fiber.
 6. The confocal microscope accordingto claim 2, further comprising a controller which controls a polarizingdirection of the polarizing direction changer according to a knowninformation of the each part on the sample.
 7. The confocal microscopeaccording to claim 1, wherein the photodetector unit includes twophotodetector units detecting an s-polarized component and a p-polarizedcomponent of the fluorescence emitted from the sample, respectively, andan calculator calculating a rotation relaxation time and a fluorescencelife based on a change of a fluorescence intensity of the s-polarizedcomponent and the p-polarized component detected with the twophotodetector units over time, respectively.
 8. The confocal microscopeaccording to claim 1, wherein the light source includes a plurality oflight sources, and each of the plurality of light sources has apolarizing direction changer changing a polarizing direction of thepolarized light beam.
 9. A confocal microscope comprising: a lightsource emitting a polarized light beam; an objective lens condensing thepolarized light beam on a sample; a rotational disk having a pluralityof pinholes or slits and leading the polarized light beam from the lightsource to the objective lens, a fluorescence image emitted from thesample being projected on the rotational disk through the objectivelens; a wavelength separator detecting a necessary wavelength band froman image passing the rotational disk; a polarization property extractorextracting a fluorescence with a predetermined polarization propertyfrom the fluorescence detected with the wavelength separator; and animaging unit imaging the fluorescence extracted by the polarizationproperty extractor.
 10. The confocal microscope according to claims 1,2, and 9, wherein the light source includes a semiconductor laser.
 11. Aconfocal microscope comprising: a light source emitting a beam; apolarizer polarizing the light beam; an optical scanner deflecting andscanning the polarized light beam; an objective lens irradiating thepolarized light beam, which is deflected and scanned by the opticalscanner, to the sample as an excitation light beam; a wavelengthseparator detecting a necessary wavelength band from a polarizedfluorescence emitted from the sample which is excited by the polarizedlight beam; and a photodetector unit having a polarization propertyextractor extracting a fluorescence with a predetermined polarizationproperty from the fluorescence detected with the wavelength separator, awavelength selector selecting a wavelength of the fluorescence extractedby the polarization property extractor, and a photodetector detectingthe fluorescence selected by the wavelength selector.